Zhijie Li

Single thalamic dopaminergic neurons project to both the neocortex and spinal cord

Cells in the rat subparafascicular thalamic nucleus (Spf) belonging to the diencephalic A11 cell group, were immunohistochemically stained with antibodies against tyrosine hydroxylase (TH) and dopamine itself. Employing a combination of retrograde fluorescent double-labeling and TH immunofluorescence techniques, we revealed the existence of dopaminergic Spf cells, giving rise to collateral projections to the neocortex and spinal cord.

Astroglial ablation prevents MPTP-induced nigrostriatal neuronal death

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a potent neurotoxin which destroys nigrostriatal dopamine neurons, resulting in irreversible idiopathic parkinsonism. MPTP displays dopaminergic neurotoxicity to humans, monkeys, cats and rodents. The oxidative conversion of MPTP to 1-methyl-4-phenylpyridine (MPP+) is responsible for the generation of its neurotoxicity. This metabolism is mediated by the action of monoamine oxidase B, which in the substantia nigra pars compacta (SNc) is localized specifically in astroglia. Employing various combinations of intra-SNc injections of MPTP and the astroglia-specific toxin, L-alpha-aminoadipic acid (L-alpha-AA), we examined the effects of selective astroglial ablation on MPTP-induced nigrostriatal neuronal death in the rat. Varying nigrostriatal cell loss was assessed primarily by the aid of fluorescent retrograde axonal tracing. Treatment with MPTP alone caused tremendous nigrostriatal cell loss, while intra-SNc co-injections of MPTP and L-alpha-AA produced protection against MPTP neurotoxicity in a dose-dependent fashion. Similar effects of L-alpha-AA occurred in the SNc pretreated with the gliotoxin just prior to or 1 day before MPTP administration. However, this preventive action by L-alpha-AA was considerably reduced 3 days after its intra-SNc injection. Interestingly, 7 days following L-alpha-AA pretreatment, nigrostriatal cell loss was even enhanced rather than attenuated by MPTP administered into the SNc. Thus, our data provide clear morphological evidence for the critical importance of the presence of astroglia in the onset of MPTP neurotoxicity.

Simple piggyBac transposon-based mammalian cell expression system for inducible protein production

Reported here is a piggyBac transposon-based expression system for the generation of doxycycline-inducible, stably transfected mammalian cell cultures for large-scale protein production. The system works with commonly used adherent and suspension-adapted mammalian cell lines and requires only a single transfection step. Moreover, the high uniform expression levels observed among clones allow for the use of stable bulk cell cultures, thereby eliminating time-consuming cloning steps. Under continuous doxycycline induction, protein expression levels have been shown to be stable for at least 2 mo in the absence of drug selection. The high efficiency of the system also allows for the generation of stable bulk cell cultures in 96-well format, a capability leading to the possibility of generating stable cell cultures for entire families of membrane or secreted proteins. Finally, we demonstrate the utility of the system through the large-scale production (140–750 mg scale) of an endoplasmic reticulum-resident fucosyltransferase and two potential anticancer protein therapeutic agents.

Collateral projection from the substantia nigra to the striatum and superior colliculus in the rat

Our retrograde fluorescent double labeling study demonstrated the existence of divergent collateral projections from the substantia nigra to the striatum and superior colliculus in the rat. These bifurcating projection neurons were located predominantly in the ventrolateral portions of the substantia nigra pars reticulata at its rostral level, where they formed a narrow band along the boundary between the substantia nigra and cerebral peduncle. Such specific projection cells were also seen in the substantia nigra pars lateralis. However, nigral neurons did not give off axonal branches to the striatum and ventromedial thalamic nucleus. The new nigral cell population proposed here might constitute a neuroanatomical substrate for abnormal saccadic eye movements clinically manifested by many parkinsonian patients.

Dopaminergic nigrotectal projection in the rat

After injecting a fluorescent tracer (Fluoro-gold) into the rat superior colliculus, retrogradely labeled neurons in the rostral, ventrolateral portions of the substantia nigra pars reticulata were also immunohistochemically labeled with tyrosine hydroxylase antisera. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) pretreatment of the medial forebrain bundle resulted in the disappearance of the nigral neurons double-labeled with the two markers. The existence of a dopaminergic nigrotectal projection susceptible to MPTP might provide a morphological substrate for abnormal saccadic eye movements in parkinsonism.

The development of laterality in the forebrain projections of midline thalamic cell groups in the rat

Bilateral forebrain (caudoputamen, nucleus accumbens and frontal cortical areas) injections of two different fluorescent retrograde tracers demonstrated that labeled cells situated in the midline nuclei of the thalamus and midbrain each project only unilaterally to the forebrain, regardless of the laterality of their perikarya. Thus, these intermingling midline perikarya send their axons primarily ipsilaterally and to a lesser degree contralaterally, but never bilaterally to the forebrain. At embryonic day 19, these midline nuclei exist as two bilaterally situated, independent structures, each projecting only ipsilaterally to the forebrain. By postnatal day 2, these perikarya fuse into a single mass on the midline. Upon fusion, many of the perikarya of the two developing subnuclei cross the midline, intermingle with each other, and thus some neurons come to have contralateral forebrain projections. These observations suggest that neurons are able to maintain their axonal projections while migrating short distances.

Recognition of EGF-like domains by the Notch-modifying O -fucosyltransferase POFUT1

Protein O-fucosyltransferase 1 (POFUT1) fucosylates the epidermal growth factor (EGF)-like domains found in cell-surface and secreted glycoproteins including Notch and its ligands. Although Notch fucosylation is critical for development, and POFUT1 deficiency leads to human disease, how this enzyme binds and catalyzes the fucosylation of its diverse EGF-like domain substrates has not been determined. Reported here is the X-ray crystal structure of mouse POFUT1 in complex with several EGF-like domains, including EGF12 and EGF26 of Notch. Overall shape complementarity, interactions with invariant atoms of the fucosylation motif and flexible segments on POFUT1 all define its EGF-like-domain binding properties. Using large-scale structural and sequence analysis, we also show that POFUT1 binds EGF-like domains of the hEGF type and that the highly correlated presence of POFUT1 and fucosylatable hEGFs has accompanied animal evolution.

Local acting Sticky-trap inhibits vascular endothelial growth factor dependent pathological angiogenesis in the eye

Current therapeutic antiangiogenic biologics used for the treatment of pathological ocular angiogenesis could have serious side effects due to their interference with normal blood vessel physiology. Here, we report the generation of novel antivascular endothelial growth factor‐A (VEGF) biologics, termed VEGF “Sticky‐traps,” with unique properties that allow for local inhibition of angiogenesis without detectable systemic side effects. Using genetic and pharmacological approaches, we demonstrated that Sticky‐traps could locally inhibit angiogenesis to at least the same extent as the original VEGF‐trap that also gains whole‐body access. Sticky‐traps did not cause systemic effects, as shown by uncompromised wound healing and normal tracheal vessel density. Moreover, if injected intravitreally, recombinant Sticky‐trap remained localized to various regions of the eye, such as the inner‐limiting membrane and ciliary body, for prolonged time periods, without gaining access either to the photoreceptors/choriocapillaris area or the circulation. These unique pharmacological characteristics of Sticky‐trap could allow for safe treatment of pathological angiogenesis in patients with diabetic retinopathy and retinopathy of pre‐maturity.

Structural basis of Notch O-glucosylation and O-xylosylation by mammalian protein-O-glucosyltransferase 1 (POGLUT1).

Protein O-glucosyltransferase 1/Rumi-mediated glucosylation of Notch epidermal growth factor-like (EGF-like) domains plays an important role in Notch signaling. Protein O-glucosyltransferase 1 shows specificity for folded EGF-like domains, it can only glycosylate serine residues in the C1XSXPC2 motif, and it possesses an uncommon dual donor substrate specificity. Using several EGF-like domains and donor substrate analogs, we have determined the structures of human Protein O-glucosyltransferase 1 substrate/product complexes that provide mechanistic insight into the basis for these properties. Notably, we show that Protein O-glucosyltransferase 1’s requirement for folded EGF-like domains also leads to its serine specificity and that two distinct local conformational states are likely responsible for its ability to transfer both glucose and xylose. We also show that Protein O-glucosyltransferase 1 possesses the potential to xylosylate a much broader range of EGF-like domain substrates than was previously thought. Finally, we show that Protein O-glucosyltransferase 1 has co-evolved with EGF-like domains of the type found in Notch.POGLUT1 is a protein-O-glucosyltransferase that transfers glucose and xylose to the EGF-like domains of Notch and other signaling receptors. Here the authors report the structure of human POGLUT1 in complexes with 3 different EGF-like domains and donor substrates and shed light on the enzyme’s substrate specificity and catalytic mechanism

Astroglial ablation by the glutamate analogue gliotoxin α-aminoadipic acid prevents l-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced nigrostriatal neuronal death

The pyridine derivative 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a potent neurotoxin which selectively destroys nigrostriatal dopamine neurons, producing neurological symptoms relevant to parkinsonism (idiopathic Parkinson’s disease). MPTP exhibits dopaminergic neurotoxicity to humans, monkeys, cats and rodents. The oxidative conversion of MPTP to 1-methyl-4-phenylpyridine (MPP+) is responsible for the generation of its neurotoxicity. This metabolism is mediated by monoamine oxidase B, which in the substantia nigra pars compacta (SNc) is localized specifically in astroglia. Employing various combinations of intra-SNc injections of MPTP and the glutamate analogue gliotoxin L-alpha-aminoadipic acid (L-α-AA), we examined both the dose- and time-dependent effects of selective astroglial ablation on MPTP-induced nigrostriatal neuronal death in the rat. Varying nigrostriatal cell loss was assessed primarily by the aid of fluorescent retrograde axonal tracing. Treatment with MPTP alone caused tremendous nigrostriatal cell loss, while intra-SNc co-injections of MPTP and L-α-AA conferred protection against MPTP neurotoxicity in a dose-dependent fashion. A similar protective action was also exerted by L-α-AA injected just prior to or 1 day before MPTP administration. However, the protective effect of L-α-AA was considerably reduced 3 days after pretreatment. Remarkably, MPTP-induced nigrostriatal cell loss was even enhanced rather than attenuated 7 days following L-α-AA pretreatment. Thus, our data provide clear morphological evidence for the critical importance of the presence of astroglia in the onset of MPTP neurotoxicity.